AmpFlSTR SGM Research Articles

Genetic analysis results of mature cystic teratomas of the ovary in Taiwan disagree with the previous origin theory of this tumor

The most accepted theory regarding mature cystic teratomas of the ovary is that they are of parthenogenetic origin from oocyte after the completion of first division. Our previous study demonstrated that the origin of mature cystic teratoma of the uterus is not related to the parthenogenetic process, but is most likely pluripotential stem cell or primordial germ cell before meiosis I. Further studies are needed to clarify the origin of benign mature cystic teratomas of the ovary in Taiwan. In the present study, we investigated the DNA profiles of 9 mature cystic teratomas of the ovary using short tandem repeat analysis with AmpFLSTR SGM Plus, Profiler PCR amplification kits. The methylation statuses of the HhaI sites in the SNRPN, H19DMR, and KvDMR regions were determined on methylation-sensitive multiplex ligation-dependent probe amplification analysis. DNA profiling data from the 9 mature cystic teratomas of the ovary excluded parthenogenetic origin, as most of the 15 short tandem repeat loci were heterozygous on genotyping. There were varying degrees of hypermethylation of SNRPN gene and KvDMR locus in the presence of maternal uniparental disomy in all 9 mature cystic teratomas of the ovary. In light of these results, we further postulated that the origin of these mature cystic teratomas of the ovary is oogonia or primary oocyte before germinal vesicle stage failure of meiosis I.

A Novel Quantitative PCR Approach Targeting Insertion/Deletion Polymorphisms (Indel-PCR) for Chimerism Quantification: Finally High Sensitivity and Quantification Capacity Together

IntroductionPost-hematopoietic stem cell transplantation (SCT) chimerism monitoring is important to assess engraftment, anticipate relapse and provide information on the development of graft versus host disease, facilitating therapeutic intervention. The aim of this study was to test the technical efficacy and clinical utility of a novel quantitative PCR approach targeting insertion/deletion polymorphisms (indel-PCR).Materials (or patients) and methodsThis study included 157 samples (81 bone marrow, 60 peripheral blood (PB), 11 T-cells, 2 myeloid cells, 2 CD34-cells, 1 NK-cells purified using immunomagnetic technology) of 24 patients who underwent SCT for haematological malignancies. Additionally, 2 sets of 11 artificial mixtures were created using PB leukocytes of two healthy subjects (a male and a female) with known percentages of male leukocytes (putative recipient): 100, 75, 50, 25, 10, 5, 3, 1, 0.1, 0.01, 0.Chimerism analysis was performed by the gold-standard STR-PCR (AmpFlSTR SGM Plus®, Life Technologies, USA) and by indel-PCR (Mentype® DIPscreen, Mentype® DIPquant, Biotype, Germany). Concordance between both methods was calculated with SPSS using intraclass correlation coefficient.ResultsThe number of informative loci identified with indel-PCR (>3/patient) was higher than with STR-PCR for all patients.Concordance between both methods for the 157 patient samples and 11 artificial mixtures was "very good" (intraclass correlation coefficient=0.96). Of note, analysis of artificial mixtures provides evidence of significantly (≥2 logs) higher sensitivity by indel-PCR (0.01%) than by STR-PCR (1%, Table 1). Moreover, indel-PCR shows unprecedented quantification capacity (Table 1). Out of the 168 samples analyzed, 32 were positive and 15 negative by both methods, while 121 were positive only by indel-PCR (95% with <1% recipient).Clinical outcome and chimerism dynamics of 24 patients are described in figure 1, data of 4 of them were censored (engraftment failure, disease progression, insufficient number of samples). All the samples presented complete donor chimerism by STR-PCR. Of the 9 patients who showed stable or decreasing percentage of recipient cells, only one (11%) presented extramedulary relapse. Of the 11 patients who presented increasing percentage of recipient cells in one determination, relapse occurred in 5 patients (45%, 3/6 patients who did not have further determinations, 2/4 who showed stable or decreasing recipient cell percentage, and 0/1 patient who presented increasing recipient cells in subsequent determinations).ConclusionThis novel indel-PCR is a simple and accurate technique that, in comparison with the current gold standard STR-PCR, shows very good concordance and provides higher rates of informative loci per patient, as well as unprecedented combined sensitivity and quantification capacity. Such features allow minutely monitoring chimerism dynamics and might improve clinical management of transplanted patients, specially predicting relapse in those patients who do not have a molecular marker available for disease follow up. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Long Allele Designations at D2S1338 and D19S433 Loci as Influenced by Various Multiplex STR Kits

European forensic laboratories are replacing the STR multiplex kits with the new generation 16/17 STR kits. This study examines the influence of the new generation kits and the new Applied Biosystems 3500xL Genetic Analyzer on the designation of long D2S1338 and D19S433 off-ladder alleles. Different allele calls were obtained using the new NGM™ (Applied Biosystems) and PowerPlex(®) ESI™ (Promega) kits compared with AmpFℓSTR(®) SGM Plus™ kit (Applied Biosystems). Sequence analysis was used to determine accurate allele designation. The new multiplex kits and the 3500xL Genetic Analyzer improved accuracy of long allele designations. DNA databases worldwide include countless profiles obtained by previous kits. Discrepancies between the new and former technologies may cause failure to detect hits. Discordance is expected due to primer sequence differences between various kits. An additional discordance, occurring in long alleles, independent of primer sequence is reported in this study.

Usefulness of the Quantitative Follow-up of Chimerism in Cell-Free Plasma DNA for the Prediction/Early Diagnosis of Complications After Hematopoietic Stem Cell Transplantation

AbstractAbstract 4146 IntroductionThe success of allogeneic stem cell transplantation (allo-SCT) relies on the result of the immune reactions between donor and recipient lymphohemopoietic systems. Chimerism quantification and monitoring post allo-SCT allows to estimate such immune reactions. In recent years, there has been a growing interest in the analysis of circulating cell-free nucleic acids in plasma. Although the ultimate origin of such nucleic acids is unknown, the most plausible hypothesis proposes cell disruption as the cause for the presence of nucleic acids in plasma. Analysis of chimerism in cell-free plasma DNA would complement the results of cell chimerism for a better prediction of different complications (engraftment failure, rejection, graft versus host disease…etc.). ObjectiveOur goal was to quantitatively monitor chimerism in cell-free plasma DNA and to evaluate its usefulness for the prediction/early diagnosis of complications after allo-SCT that would favour early therapeutic intervention. Patients and MethodsThe study comprised 270 plasma samples from 29 allo-SCT patients. With a median follow up of 45,8 months (14,6– 74,7).Whole blood (WB) (10 mL) was collected in EDTA containing tubes and was processed immediately after collection. Plasma was separated from the blood cells by centrifugation at 1600g for 10 min and cell-free plasma DNA was extracted according to the blood and body fluid protocol of the QIAamp DNA Blood Mini Kit (Qiagen). Quantitative chimerism analysis was performed by microsatellite PCR (STR; AmpFlSTR SGM plus, Applied Biosystems) in whole blood (WB), T-cells (CD3+) purified using immunomagnetic means (Miltenyi Biotec) and cell-free plasma DNA.Chimerism was monitored at regular intervals post-transplant (+30,+60, +90, +180, +365). Sensitivity of the technique is 1% in WB and cell-free plasma DNA, and 5% in T-cells (derived from a 95% purity of enriched samples). ResultsDuring the first year of allo-SCT, the number of patients with mixed chimerism in cell-free plasma DNA was higher than in WB and T-cells (Table 1). Cell-free plasma DNA mixed chimerism was observed for a longer timer after transplant than “cellular” mixed chimerism, with a median of 105 vs. 40 and 27 days for cell-free plasma DNA, T-cells and WB, respectively.Chimerism analysis revealed two groups of patients, one with mixed chimerism in both cell-free plasma DNA and “cellular DNA”, in which the percentage of recipient plasma DNA ranged between 10–30%, probably mostly derived from hematopoietic cells. The other group presented mixed chimerism in cell-free plasma DNA but complete “cellular” chimerism (100% donor) in which cells the percentage of recipient plasma DNA was lower than in the former group (1–10%).A greater number of patients with graft versus host disease (GVHD),mainly acute but also chronic, presented mixed chimerism in cell-free plasma DNA and complete chimerism in WB and T-cells (Table 2). The presence of recipient cell-free plasma DNA could be derived by cell disruption in GVHD target organs. DiscussionQuantitative follow-up of chimerism in cell-free plasma DNA, does not reproduce the results of “cellular” chimerism, as reported previously, and therefore, could not replace it for post-SCT follow-up. On the other hand, chimerism analysis in cell-free plasma DNA could be combined with the conventional follow-up of “cellular” chimerism for an improved diagnosis of life-threatening complications such as acute and chronic GVHD.Table 1Percentage of patients in mixed chimerism during the first year post-allo-SCT and time to CC in whole blood, T-cells and cell free plasma DNA.Days post-allo-SCTDays until CC (median)+30+60+90+180+365WB3524204027T cells48282011040Plasma8372662410105Table 2Pacients with mixed chimerism in whole bllod, T-cells and cell free plasma DNA in the moment of aGVHD and cGVHD developed.acute GVHDchronic GVHDTotal15/29 (51%)15/29 (51%)WB2/15 (13,3%)0/15 (0%)T cells4/15 (26,6%)2/15 (13,3%)Plasma11/15 (73,4%)7/12 (58,3%) Disclosures:No relevant conflicts of interest to declare.

A Search for Obligatory Paternal Alleles in a DNA Database to Find an Alleged Rapist in a Fatherless Paternity Case

A sexual assault case resulted in a pregnancy, which was subsequently aborted. The alleged father of the fetus was unknown. Maternal and fetal types were obtained using the 11-locus AmpFℓSTR(®) SGM Plus(®) kit. The national DNA database was searched for the paternal obligatory alleles and detected two suspects who could not be excluded as father of the male fetus. Additional typing using the AmpFℓSTR(®) Minifiler(™) kit, containing three additional autosomal loci, was not sufficient to exclude either suspect. Subsequent typing using the PowerPlex(®) 16, containing four additional loci, and Y-Filer(™) kits resulted in excluding one suspect. Searching a database for paternal obligatory alleles can be fruitful, but is fraught with possible false positive results so that finding a match must be taken as only preliminary evidence.

Genetic structure of Moroccan population using 15 STRs of NGM kit

Polymerase chain reaction (PCR) amplification using the AmpFℓSTR ® NGM™ Kit (“NGM,” an acronym for “Next Generation Multiplex”) was performed in random sample of 180 unrelated individuals from three Moroccan population groups (Arab Speaking, Berber Speaking, and Sahrawi). Allele frequency and other forensically relevant statistics data were generated for the NGM™ multiplex kit includes the original 10 STR loci from the AmpFℓSTR SGM Plus ® kit (D3S1358, vWA, D16S539, D2S1338, D8S1179, D19S433, TH01, FGA, D21S11, D18S51) together with five additional STRs (D10S1248, D22S1045, D2S441, D1S1656 and D12S391) and the Amelogenin sex-determination locus. Population study was conducted to evaluate usefulness of the loci (especially the five new microsatellite systems) in forensic genetic identification examinations. All Fifteen autosomal STR loci were found to be in Hardy–Weinberg equilibrium. Discrimination power was particularly high in case of D18S51 and D12S391 STR loci.

Applying a PCR inhibitor tolerant DNA polymerase blend in forensic DNA profiling

Crime scene samples often contain extraneous compounds that may interfere with PCR-based DNA analysis, resulting in imbalanced, partial or blank/negative DNA profiles. Customising the chemical content of the PCR reaction is a strategy that may increase PCR inhibitor tolerance without manipulating the sample. We have validated a modified version of Amp FlSTR SGM Plus, replacing Ampli Taq Gold DNA polymerase with a customised blend of two alternative polymerases, Ex Taq Hot Start and PicoMaxx High Fidelity. Allele calls are identical to standard analysis. Stutter sizes and balance values are indistinguishable. The modified chemistry provides increased resistance to PCR inhibitors, resulting in an elevated number of detected alleles for various problematic crime scene samples.

Designing Polymerase Chain Reaction (PCR) Primer Multiplexes in the Forensic Laboratory

The polymerase chain reaction (PCR) is a common experiment in upper-level undergraduate biochemistry, molecular biology, and forensic laboratory courses as reagents and thermocyclers have become more affordable for institutions. Typically, instructors design PCR primers to amplify the region of interest and the students prepare their samples for PCR and analyze the results. However, primers can also be designed in undergraduate laboratories with students at this level. In a course that focuses on forensic DNA molecular biology for forensic chemistry students, students have used the Applied Biosystems AmpFlSTR SGM Plus kit that amplifies DNA at eleven regions in a single test tube. It is important for forensic chemistry students to be able to design and analyze a single set of primers and, more importantly, create multiplexes of primers. This enables students to more fully understand how the primers and the kits that are routinely employed by the crime laboratories function. Creating a single set of primers does not demonstrate the extent of design and engineering inherent in creating multiplexes or adequately prepare students for research and careers in the field. The in silico method described herein uses free bioinformatics tools and results in student-designed multiplexes for Combined DNA Index System (CODIS) loci. Sample student data are shown.

A comparison of methods for forensic DNA extraction: Chelex-100® and the QIAGEN DNA Investigator Kit (manual and automated)

Efficient isolation of DNA from a sample is the basis for successful forensic DNA profiling. There are many DNA extraction methods available and they vary in their ability to efficiently extract the DNA; as well as in processing time, operator intervention, contamination risk and ease of use. In recent years, automated robots have been made available which speed up processing time and decrease the amount of operator input. This project was set up to investigate the efficiency of three DNA extraction methods, two manual (Chelex®-100 and the QIAGEN DNA Investigator Kit) and one automated (QIAcube), using both buccal cells and blood stains as the DNA source. Extracted DNA was quantified using real-time PCR in order to assess the amount of DNA present in each sample. Selected samples were then amplified using AmpFlSTR SGM Plus amplification kit. The results suggested that there was no statistical difference between results gained for the different methods investigated, but the automated QIAcube robot made sample processing much simpler and quicker without introducing DNA contamination.

Dynamics of Chimerism In Regulatory T Lymphocytes (Treg; CD4+/CD25+) After Allogeneic Stem Cell Transplantation

AbstractAbstract 1328 Introduction:CD4+CD25+ regulatory T-cells (Treg) play an important role in inducing and maintaining allogeneic tolerance and can inhibit graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (SCT). However, the dynamics of donor and recipient Treg cell populations after Allo-SCT has not been studied yet. Objective:To analyze the dynamics of chimerism in Tregs and compare it to that of T lymphocytes (TL; CD3+) and whole blood leukocytes (WBL). Material and Methods:The study includes 53 patients subjected to Allo-SCT (myeloablative and non-myeloablative). PB samples were obtained weekly during the first month and every 14 days after day +30 until complete chimerism (CC) was achieved, and at fixed time-points (+30, +60, +90, +180, +365) thereafter. TL and Tregs were purified from PB until CC was achieved using inmunomagnetic technology (Miltenyi Biotec; CD3+ Microbeads and CD4+/CD25+ Treg Isolation Kit, respectively). Chimerism analysis was performed by microsatellite PCR (STR-PCR; AmpFlSTR SGM Plus; Applied Biosystems) on genomic DNA obtained from WBL as well as on cell lysates obtained from purified TL and Tregs. Complete chimerism was considered in samples with percentage of recipient cells <1% (sensitivity of the STR-PCR) for WBL samples and <5% (minimum purity of 95% as estimated by flow cytometry) for purified cell lineages. Results:Median follow up for the whole cohort of patients was 338 days. CC was spontaneously achieved in WBL, TL and Tregs in 45/53 patients (85%) in a median time of 35.41, 38.8 and 42.5 days respectively. 5/3 patients (9%) suffered from graft failure/rejection showing mixed chimerism (MC) with increasing percentages of recipient cells both in WBL and leukocyte lineages. 3/53 patients (6%) maintained mixed chimerism (MC) in WBL and leukocyte lineages at day +180, with no signs of graft rejection or disease relapse. Analysis of the chimerism dynamics of those patients who spontaneously achieve CC revealed two different groups: Group 1 included 25/45 patients who achieved CC at the same time in WBL, TL and Tregs. Group 2 included 20/45 patients who achieved CC in WBL while maintaining MC in TL and Tregs. Interestingly enough, 9/20 patients from Group 2 maintained MC in Tregs 7–75 days after achieving CC in both WBL and TL (Figure 1). In a preliminary analysis, the small sample size precluded from obtaining statistically significant associations between the dynamics of chimerism in Tregs and the development of complications such as relapse or GVHD after SCT. [Display omitted] Conclusions:To our knowledge, this is the first study dealing with Treg chimerism after SCT. We have shown it is feasible and can be performed on a routine basis together with standard lineage specific chimerism follow up. Although there is an association between chimerism dynamics in Tregs and TL, this is not absolute and a percentage of patients maintain residual Tregs of recipient origin after WTL and TL have become of complete donor origin. In this small cohort, Treg chimerism did not influence the development of post-SCT complications. Analysis of a larger and more homogeneous cohort would allow establishing the usefulness of Treg chimerism testing for the management of transplanted patients. Disclosures:No relevant conflicts of interest to declare.

Effects of Histological Staining on the Analysis of Human DNA from Archived Slides*

Archived slides of cell smears treated with histological stains for sperm detection are often the only source of DNA available when cold cases are reopened. There have been conflicting reports as to the negative effects of particular histological stains on DNA recovery and quality from human cells, making stain selection an important consideration for forensic laboratories. This study investigates the effect of several staining systems on DNA recovery from histological slide samples stored from 0 to 10 weeks. DNA profiles obtained after analysis of these samples with AmpFlSTR(®) Identifiler™ and increased cycle AmpFlSTR(®) SGM Plus™ short tandem repeat (STR) profiling systems and the effects that these stains have on DNA quantity and quality over time are described. Results indicate that Christmas Tree and Hematoxylin and Eosin stains do not have significantly different effects on DNA quality after 10-week storage of slides. This research will assist scientists to select staining systems that have minimal deleterious effects on the DNA recovered.

Population genetic data for 17 STR markers from Lebanon

Seventeen autosomal STRs were analyzed (D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, CSF1PO, FGA, TH01, TPOX, vWA, Penta D, and Penta E) in the Lebanese population. A total of 192 unrelated individuals were genotyped for the 15 autosomal STRs in the Promega PowerPlex 16 STR kit. An additional 275 unrelated individuals were genotyped for the Applied Biosystems AmpFlSTR Identifiler and SGM + STR kits. Allele frequencies for the shared CODIS 13 loci among the three STR kits tested were not significantly different among individuals within the Lebanese population. Forensic and population genetic parameters for the 17 loci were calculated. We also compared the allele frequencies from this population with other populations in the same geographic vicinity.

The ANALYSIS of Chimerism IN ACTIVATED T Lymphocites CD25+ Improves the CAPACITY of the T Lymphocytes CD3+ Chimerism Dinamic to Predict COMPLICATIONS AFTER Allogenic STEM CELL TRANSPLANTATION.

Abstract 4307 IntroductionComplications after Allogenic Stem Cell Transplantation (Allo-SCT) are influenced by the immunological fenomena associated to the alloreactivity/allotolerance between donor and recepient. In this context, the usefulness of chimerism quantification in T lymphocytes for the prediction of complications after Allo-SCT is well known. However, to our knowledge, there is no information on the utility of chimerism analysis in activated lymphocytes (CD25+). ObjectiveTo stablish an asociation betwen the more common complications post Allo-Tph (aGVHD, cGVHD, relapse, reject), and the chimerism dynamics in CD25+ activated lymphocytes in the early period post-transplantation, trying to predict the patients with more risk of suffering from this complications. Materials and MethodsThe study included 38 Allo-SCT (17 NMA, 21 MA; 7 Haploidentical, 13 non-related donor, 18 identical donor; 7 UCB, 5 BM, 26 PB). Chimerism analysis was performed every 2 weeks until complete chimerism (CC) was achieved, and every 3 months thereafter. Follow up was censored when patients received DLI or 2nd Allo-SCT.Chimerism quantification was performed by microsatellite PCR (STR-PCR; AmpFlSTR SGM Plus; Applied Biosystems) on DNA obtained from peripheral blood and purified cell lineages (95% putity) by immunomagnetic technology (Miltenyi Biotec). ResultsTable 1 shows the results at day +30 for T and activated lymphocytes (CD3+ and CD25+) allocating patients in two groups depending on the relative percentage of recipient cells (% rec) in both cell lineages. 20 patients were in CC in CD25+, maintening this CC during the study. 18 patients were found in MC in CD25+ (18/38 MC). 1/18 was in CC in CD3+ TL, and 10/18 were in CC in whole blood. 17/18 were in MC both in CD3+ and CD25+ lymphocytes,Table 1Day + 30 post Allo-Tph n=17% rec CD25 >% rec CD3% rec CD3>% rec CD25Patients125aGVHD >15 (41%)1 (20%)cGVHD4 (33%)1 (20%)CQ day+907 (58%)5 (100%)Relapse4 (33%)0 (0%)Rejection1 (8%)0 (0%)Although no statistical significance is achieved due to the reduced sample size, patients in the first group (% rec higher in activated that in T lymphocytes) tend to achieve CC later and to show higher risk of developing complications such as acute and chronic GVHD, relapse or rejection. ConclusionsA higher incidence of complications post Allo-SCT (aGVHD, cGVHD, relapse, rejection) in patients with % of recipient in CD25+> % recipient in CD3 at day 30. Chimerism analysis in activated lymphocytes (CD25+) is of great utility after Allo-SCT since it allows beter prediction of complications than standard follow up of whole blood or T lymphocytes. Disclosures:No relevant conflicts of interest to declare.

Improved Forensic DNA Analysis Through the use of Alternative DNA Polymerases and Statistical Modeling of DNA Profiles

DNA evidence, linking perpetrators to crime scenes, is central to many legal proceedings. However, DNA samples from crime scenes often contain PCR-inhibitory substances, which may generate blank or incomplete DNA profiles. Extensive DNA purification can be required to rid the sample of these inhibitors, although these procedures increase the risk of DNA loss. Most forensic laboratories use commercial DNA amplification kits (e.g., AmpFlSTR SGM Plus) with the DNA polymerase AmpliTaq Gold as the gold standard. Here, we show that alternative DNA polymerase-buffer systems can improve the quality of forensic DNA analysis and efficiently circumvent PCR inhibition in crime scene samples, without additional sample preparation. DNA profiles from 20 of 32 totally or partially inhibited crime scene saliva samples were significantly improved using Bio-X-Act Short, ExTaq Hot Start, or PicoMaxx High Fidelity instead of AmpliTaq Gold. A statistical model for unbiased quality control of forensic DNA profiles was developed to quantify the results. Our study demonstrates the importance of adjusting the chemistry of the PCR to enhance forensic DNA analysis and diagnostic PCR, providing an alternative to laborious sample preparation protocols.

Developing STR databases on structured populations: The native South Siberian population versus the Russian population

Developing a forensic DNA database on a population that consists of local ethnic groups separated by physical and cultural barriers is questionable as it can be genetically subdivided. On the other side, small sizes of ethnic groups, especially in alpine regions where they are sub-structured further into small villages, prevent collecting a large sample from each ethnic group. For such situations, we suggest to obtain both a total population database on allele frequencies across ethnic groups and a list of θ-values between the groups and the total data. We have genotyped 558 individuals from the native population of South Siberia, consisting of nine ethnic groups, at 17 autosomal STR loci of the kit packages AmpF lSTR SGM Plus и AmpF lSTR Profiler Plus. The groups differentiate from each other with average θ-values of around 1.1%, and some reach up to three to four percent at certain loci. There exists between-village differentiation as well. Therefore, a database for the population of South Siberia is composed of data on allele frequencies in the pool of ethnic groups and data on θ-values that indicate variation in allele frequencies across the groups. Comparison to additional data on northeastern Asia (the Chukchi and Koryak) shows that differentiation in allele frequencies among small groups that are separated by large geographic distance can be even greater. In contrast, populations of Russians that live in large cities of the European part of Russia are homogeneous in allele frequencies, despite large geographic distance between them, and thus can be described by a database on allele frequencies alone, without any specific information on θ-values.

Development and Validation of the AmpFℓSTR® MiniFilerTM PCR Amplification Kit: A MiniSTR Multiplex for the Analysis of Degraded and/or PCR Inhibited DNA*

DNA typing of degraded DNA samples can be a challenging task when using the current commercially available multiplex short tandem repeat (STR) analysis kits. However, the ability to type degraded DNA specimens improves by redesigning current STR marker amplicons such that smaller sized polymerase chain reaction (PCR) products are generated. In an effort to increase the amount of information derived from these types of DNA samples, the AmpFlSTR MiniFiler PCR Amplification Kit has been developed. The kit contains reagents for the amplification of eight miniSTRs which are the largest sized loci in the AmpFlSTR Identifiler PCR Amplification Kit (D7S820, D13S317, D16S539, D21S11, D2S1338, D18S51, CSF1PO, and FGA). Five of these STR loci (D16S539, D21S11, D2S1338, D18S51, and FGA) also are some of the largest loci in the AmpFlSTR SGM Plus kit. This informative nine-locus multiplex, which includes the gender-identification locus Amelogenin, has been validated according to the FBI/National Standards and SWGDAM guidelines. Our results demonstrate significant performance improvements in models of DNA degradation, PCR inhibition, and nonprobative samples when compared to the AmpFlSTR Identifiler and SGM Plus kits. These data support that the MiniFiler kit will increase the likelihood of obtaining additional STR information from forensic samples in situations in which standard STR chemistries fail to produce complete profiles.

Identification of variant alleles at AmpFlSTR SGM Plus STR loci in a sample population of Bangladesh

DNA profiles obtained from 442 individuals using AmpFlSTR® SGM Plus™ PCR amplification kit (Applied Biosystems, Foster City, CA, USA) were screened for variant alleles not included within allelic ladder provided by the manufacturer. A total of 5 distinct variants were identified at 3 of the 10 European core STR loci tested. This study identified one variant at D16S539 locus (allele 7), two variants at D21S11 locus (allele 29.3 and 34.1) and two other rare variants at D19S433 locus (allele 11.2 and 18). Of these 5 alleles, variant 29.3 and 34.1 found at D21S11 have been previously reported in Caucasians and African Americans. The rest three variants (allele 7 at D16S539, allele 11.2 and 18 at D19S433) have been separately reported in STRBase (http://www.cstl.nist.gov/biotech/strbase), but no published reports are available. All these alleles fell within the allelic range of the allelic ladder except allele 18 at D19S433 locus, which fell outside the range.

Detectability of SGM Plus profiles in heart and lungs tissue samples incubated in different environments

The aim of the study was assessment of environmental effect on typeability of AmpFlSTR SGM Plus loci: D3S1358, VWA, D16S539, D2S1338, D81179, D21S11, D18S51, D19S433, TH01, FGA and gender marker amelogenin. Heart and lungs specimens collected during autopsies of five persons aged 20–30 years were incubated at 21 °C and 4 °C in different environmental conditions, fresh different water and soil conditions. DNA was extracted by organic method from tissue samples collected in 7-day intervals and subsequently typed using AmpFlSTR SGM Plus kit and ABI 310. Incubation at 21 °C and prevented air access, as well as in peat soil and in sand favoured faster DNA degradation reflected by decrease in typeability rate. In samples with negative genotyping results no DNA was found by fluorometric quantitation. Decomposed soft tissues are potential material for DNA typing.

The variation of 15 autosomal microsatellite DNA loci in five indigenous populations of South Siberia

The allele frequencies of 15 autosomal STR loci (D3S1358, vWA , FGA , TH01 , TPOX , CSF1PO , D5S818, D13S317, D7S820, D16S539, D2S1338, D8S1179, D21S11, D18S51, and D19S433) included into the AmpFlSTR SGM Plus and AmpFlSTR Profiler Plus kits (Applied Biosystems, United States) were deter- mined for five indigenous populations of South Siberia: Buryats, Altaians, Tofalars, Sojots, and Khakassians ( N = 261). No significant differences in allele frequencies were found between the populations. The combined power of discrimination of the STR loci was determined for every population. DOI: 10.1134/S0026893307040024

Typeability of AmpFlSTR SGM Plus Loci in Brain and Thyroid Gland Tissue Samples Incubated in Different Environments*

Autolysis and putrefaction are crucial factors responsible for degradation of cells, tissues, and organs. Postmortem changes may assume different course depending on extrinsic and intrinsic conditions. The aim of the study was assessment of environmental effect on typeability of AmpFlSTR SGM Plus loci: D3S1358, VWA, D16S539, D2S1338, D81179, D21S11, D18S51, D19S433, TH01, FGA, and gender marker amelogenin. Brain and thyroid gland tissue specimens collected during autopsies of five persons aged 20-30 years were incubated at 21 degrees C and 4 degrees C in different environmental conditions. DNA was extracted by organic method from tissue samples collected in 7-day intervals and subsequently typed using AmpFlSTR SGM Plus kit and ABI 310. A fast decrease in typeability rate was seen in specimens incubated in peat soil and in sand. Brain tissue samples were typeable in all AmpFlSTR SGM Plus loci within 126 days of incubation at 4 degrees C. Faster DNA degradation was recorded in thyroid gland specimens. In samples with negative genotyping results, no DNA was found by fluorometric quantitiation.